We have developed a cellular model of microvillus inclusion disease. Microvillus inclusion disease (MVID or MID~ MIM#251850) is characterized by the loss of microvilli and the appearance of microvillus inclusion bodies in intestinal epithelial cells. This results in a lss of intestinal absorption, rapidly leading to death without parenteral nutrition (TPN). Te only documented gene involved in this disease in the gene encoding the molecular motor, myosin Vb. The majority of the mutations result in loss of functional myosin Vb protein, while a subset of the mutations are missense mutations that likely alter motor function. We have generated a line of intestinal epithelial cells (Caco-2 cells) in which myosin Vb levels are <5% of wild type and can no longer maintain microvilli when they are polarized. This will serve as a cell culture model for a subset of microvillus inclusion disease, and can be used as a cellular assay for myosin Vb function. We will characterize the impact of MVID-causing missense mutations on the kinetics of the myosin myosin Vb motor. Based on the outcome of the biochemical characterizations, we will create a number of new lines of Caco-2 cells that express representative mutant myosin Vb proteins in the near null (knock down) background, thus creating new cellular models of MVID. Upon differentiation of these cells lines, we will examine the impact the myosin Vb missense mutations on microvilli development/maintenance. The creation of these lines will set the stage for future studies to understand the role of myosin Vb in apical membrane recycling as well as provide a better understanding of the pathogenesis of MVID.